We have recently demonstrated the expression of a gene, the ubiquitin E3 ligase, GRAIL, in CD4+ T cells during the induction and maintenance phases of anergy in vivo, following our original description of the ability of over expression of GRAIL in vitro to block activation induced IL-2 transcription in T cell hybridomas. Additionally, however, expression of GRAIL in naive CD4+ TCR transgenic T cells in vivo, following retroviral transduction of bone marrow cells and adoptive transfer into immuno-incompetent recipients, rendered resultant peripheral CD4+ T cells refractory to antigen stimulation, both for proliferation and IL-2 production, resembling the typical phenotype of anergic CD4+ T cells. The striking finding that expression of an enzymatically inactive version of the E3 ligase, H2N2 GRAIL, effectively prevented the establishment of the anergy phenotype in similar bone marrow transduced adoptively transferred peripheral CD4+ T cells, clearly establishes the importance of this new E3 ligase as a central player in the anergy phenotype. Studies in this competing renewal proposal will take advantage of these data to further explore the function and regulation of GRAIL in anergy, not only in models in vitro, but will also look into the potential role of GRAIL and its binding partners, DOG and SOG, in animal models of cancer and autoimmunity as well as an attempt to establish a role for these proteins (or their dysregulation) in human disease. These studies will be accomplished under two specific aims: (1) Molecular and biochemical characterization of the GRAIL pathway in anergic T cells; and (2) Analysis of GRAIL and GRAIL associated protein expression in animal models of cancer and autoimmunity and in human disease counterparts. Although many of the techniques that will be used in these studies are commonly practiced, innovative methods of screening for E3 ligase substrates, techniques to follow GRAIL expressing cells in vivo using knock-in technology, and the development of a novel lentiviral vector for bone marrow hematopoietic stem cell transduction and reconstitution of immuno-incompetent mice are novel techniques that have recently been or are being developed in the lab. Dysregulation of GRAIL and/or its binding partners, DOG or SOG, in human diseases of immune or cellular dysfunction, is suggested by preliminary data. If this proves correct, it will open studies of an exciting pathway of cellular signaling whose dysregulation has important implications in cancer and autoimmunity.